Study setting, design, and bacterial isolates

The prospective study was conducted in the Department of Microbiology and Multidisciplinary Research Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. The study included 60 isolates of previously confirmed and characterized CRAB. Of these 30 were pathogens from clinically confirmed infections [12] and 30 were colonizers recovered from the hospital environment and hands of healthcare workers from the adult ICU [11]. The source of these isolates has been shown in Supplementary Table 1.

Phenotypic determination of virulence factorsBiofilm formation

The capacity of biofilm formation in pathogenic CRAB and colonizer CRAB isolates from the environment was tested by the standard 96-well microtiter plate assay with 0.1% crystal violet staining method as previously described [13, 14]. The bacterial suspension was prepared by adding a pure isolated colony of A. baumannii into LB (Luria Bertani) broth medium. After overnight incubation at 37ºC the bacterial suspension was diluted 1:20 in LB broth in order to match 0.5 McFarland (∼1.5 × 108 CFU/mL). The optical density measurement at 578 nm (OD578) was taken using LisaScan® EM micro-titer plate reader (Eli-LIMS, Transasia Bio Medical Ltd., India). The categories of no/weak/moderate/strong biofilm producers were selected on the basis of three standard deviations above the mean OD (0.054) of a clean microtiter plate stained by the same procedure [13, 14]. Biofilm formation in Staphylococcus epidermidis ATCC 35,984 was used as a positive control.

Desiccation survival

The tolerance to survive desiccation between the pathogenic CRAB and colonizer CRAB isolates from the environment was tested for more than 100 days. Briefly, 1 ml of overnight grown culture was centrifuged at 15,000 rpm for 5 min. The pellets were twice washed with sterile distilled water and subsequently resuspended in 1 ml of distilled water. A volume of 20 µl of this suspension was placed onto the cover slips and stored inside covered Petri plates. These Petri plates were kept within sterile plastic boxes with relative humidity maintained at 31% ± 3% inside the plastic boxes. Viable counts were determined at every 20 days’ interval [13,14,15].

Hemolytic activity

The hemolytic activity of these isolates was tested as described by Boone RL et al., 2021. Briefly, the isolates were streaked over Columbia 5% sheep blood agar plates (HiMedia Laboratories Pvt Ltd, India) and incubated overnight at 37 °C. The 2–3 pure isolated colonies were subcultured into 5 ml LB broth (HiMedia Laboratories Pvt Ltd, India) and kept for overnight incubation at 37°C with constant shaking at 180 rpm. The bacterial suspension was adjusted according to 0.5 McFarland standard and 3 µl of this suspension was inoculated on fresh Columbia 5% sheep blood agar plates. The plates were kept for 24 h incubation at 37°C and further observed for ß-hemolysis [5].

Motility testing Surface-associated motility

To determine the surface-associated motility, swimming agar plates (peptone 5 g/L, NaCL 2.5 g/L and 0.3% agarose) were prepared. The overnight grown bacterial suspension in LB broth was normalized with 0.5 McFarland and 2 µl was inoculated in the center of the plate. Plates were kept for incubation at 37 °C in dark. The migration from the center was noted at 24 h post-inoculation [5].

Twitching motility

For determination of twitching motility, plates containing peptone 10 g/L, NaCl 5 g/L and 1% agarose were prepared. A single pure isolated colony from overnight growth was stabbed at the agarose/petriplate interphase with the help of a sterile straight wire. The inoculated plates were kept for incubation at 37 °C in dark. The result was interpreted at 24 h post-inoculation with 0.1% staining. For this, the agarose was carefully removed from the plates and the plates were subsequently washed thrice with phosphate buffered saline (PBS). The plates were stained with 0.1% crystal violet for 5 min and again washed gently with PBS. The plates were kept at room temperature for air dry and the migration was measured with the help of a scale. The plates were freshly prepared on the day of inoculation. A. baumannii ATCC 19,606 was used as control strain as it exhibits very low motility (mean motility diameter of 1.1 cm) [5].

Molecular detection of virulence genes

The presence of following genes; pgaA, csuE, bap, ompA, abaI, pilA, bauA in all the isolates was detected by conventional Polymerase Chain Reaction (PCR) (T100 PCR thermal cycler, Bio-Rad laboratories India Pvt. Ltd ) as described earlier [8, 16, 17]. The oligonucleotide sequences used as primers have been shown in Table 1. Reaction mixture (25 µL) was prepared by adding 2.5 µL Taq DNA buffer, 2 µL of dNTP, and 1 µL of each primer (10 picomole; Eurofins Scientific), 0.3 µL of Taq DNA polymerase (Genei, Bangalore, India). To maintain volume, 5 µL of template DNA (100 ng/mL) and nuclease free water was added. Reactions were run under the following conditions: for bap, ompA and csuE genes, initial denaturation 94ºC for 3 min, 35 cycles of 94 °C for 30 s, 56ºC for 30 s, 72ºC for 30 s. and final extension at 72ºC for 10 min.For abaI and ompA genes, initial denaturation 94ºC for 3 min, 35 cycles of 94 °C for 30 s, 59ºC for 30 s, 72ºC for 30 s. and final extension at 72ºC for 10 min. For pilA and bauA genes, initial denaturation 95ºC for 10 min, 40 cycles of 95 °C for 15 s, 60ºC for 60 s, 72ºC for 30 s. and final extension at 72ºC for 10 min.The amplified PCR products were identified in 2% agarose gel by agarose gel electrophoresis.

Table 1 Primer sequences used for the amplification of the target genesExpression of virulence genes by quantitative real-time PCR (qRT-PCR)

Based on the result of phenotypic tests and presence/absence of virulence genes, 10 isolates of each pathogenic CRAB and colonizer CRAB were included in the expression study. Total RNA was extract using RNeasy Mini Kit (Qiagen, Pvt. Ltd, India) according to manufacturer’s instruction form the freshly grown culture in LB broth. cDNA was prepared by the reverse transcriptions of 400ng/µl of total RNA using oligo dT primers and RevertAid transcriptase in a total reaction volume of 20 µl (ThermoScientific, Pvt. Ltd, India). qRT-PCR was performed using SYBR Green PCR master mix (GCC Biotech, India Pvt. Ltd.) and primers described in Table 1.Each reaction mixture was prepared in a final volume of 20 µl containing 10 µl of SYBR Green PCR master, 0.5 µl of each forward and reverse primer (10 picomole; Eurofins Scientific, India), 5 µl of 400ng cDNA and remaining volume was maintained by adding nuclease free water. The reaction was run under following condition: initial denaturation 95ºC for 5 min, 39 cycles of 95ºC for 15 s, 60ºC for 20 s, 72ºC for 3 min.16 S rRNA gene was used for normalization of gene expression. A. baumannii ATCC 19,606 used asreference strain. Fold change in gene expression were calculated using comparative Ct method (2¯ΔΔCt ).

Galleria mellonella survival assay

The G. mellonella (wax moth) were obtained from UDeS Honey Farms, Varanasi, India and reared in the laboratory at 30 °C in dark with natural beeswax diet. The last instar larvae, weighing 250 mg – 350 mg were used for the experiment. For survival assay a total of 10 isolates of each pathogenic CRAB and colonizer CRAB included in expression analysis were selected. Bacterial suspension was prepared by mixing 2–3 pure isolated colonies into sterile PBS, from overnight growth on MacConkey agar plates. The bacterial cell count was adjusted to 1.5 × 108 CFU/ml according 0.5 McFarland. 5 µl of the bacterial suspension was injected into the last proleg of the larvae with help of microliter™ syringe (10 µl glass syringe FN (701 N) P/n 80300, Hamilton® Reno, Nevada, USA). A set of control group i.e., larvae injected with A. baumannii ATCC 19,606 bacterial suspension, injected with sterile PBS and free larvae without any injection were included with each experiment. The larvae were kept at 37 °C in dark and observed every 24 h for consecutive 6 days. The survival of larvae was assessed by any response to physical stimuli. The experiments were repeated two times with 5 larvae in each experimental group [18].

Statistical analysis

The degree of biofilm formation and surface associated motility and expressional changes between the pathogenic CRAB and colonizer CRAB from the environment was statistically compared using t-test. The in-vitro and in-vivo survival proportion between both the groups was compared with the help of Log-rank (Mantel-Cox) test. All the analysis was done using GraphPad prism version 5 Software: La Jolla California USA, and using the data derived from at least two biological replicates.